Toxic PARP trapping upon cAMP-induced DNA damage reinstates the efficacy of endocrine therapy and CDK4/6 inhibitors in treatment-refractory ER+ breast cancer

Resistance to endocrine therapy and CDK4/6 inhibitors, the standard of care (SOC) in estrogen receptor-positive (ER+) breast cancer, greatly reduces patient survival. Therefore, elucidating the mechanisms of sensitivity and resistance to SOC therapy and identifying actionable targets are urgently needed. Here, we show that SOC therapy causes DNA damage and toxic PARP1 trapping upon generation of a functional BRCAness (i.e., BRCA1/2 deficiency) phenotype, leading to increased histone parylation and reduced H3K9 acetylation, resulting in transcriptional blockage and cell death. Mechanistically, SOC therapy downregulates phosphodiesterase 4D (PDE4D), a novel ER target gene in a feedforward loop with ER, resulting in increased cAMP, PKA-dependent phosphorylation of mitochondrial COXIV-I, ROS generation and DNA damage. However, during SOC resistance, an ER-to-EGFR switch induces PDE4D overexpression via c-Jun. Notably, combining SOC with inhibitors of PDE4D, EGFR or PARP1 overcomes SOC resistance irrespective of the BRCA1/2 status, providing actionable targets for restoring SOC efficacy.


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All studies must disclose on these points even the disclosure is negative.During patient sample analysis, few of the TMA cores that have fallen off the slide during slide preparation were not stained and not included.
For in vitro experiments, replicates that were identified as outliers by Grubbs method (alpha=0.1)using GraphPad software were excluded.
Experiments were repeated at least twice and reproducible results were obtained.
In vitro samples were randomly allocated into different treatment groups.For in vivo experiments, mice were randomly allocated into different treatment groups.
Investigators were not blinded while allocating mice into groups, during treatment, data collection or analysis because the sample names contained treatment information.
the accession number GSE932042.Data presented on Figs 2m-p were generated by analyzing the data from GSE874112.Data presented on Fig 4e was generated by analyzing the data from GSE81538.Data presented on Figs 5a, b were generated by analyzing the data from GSE124647.Data presented on Figs 6dwas generated by analyzing the data from GSE81538.Data presented on Supplementary Fig.S4cwas generated by analyzing the data from GSE202203.These GEO dataset are available at GEO depository (https://www.ncbi.nlm.nih.gov/geo/).All patients were ER-positive and 85%were also PR-positive.The patients were diagnosed with Grade I (6.3%),GradeII (50.7%), and Grade III (43.1%).The patients were diagnosed with Stage I (17.8%),StageII (56.8%), and Stage III (25.4%).All patients were treated with endocrine therapy with or without radiotherapy and chemotherapy.Tumors from 172 ER+ breast cancer patients that were diagnosed between 2000 and 2016 at Hacettepe University School of Medicine, Ankara, Turkey were analyzed for this study.De-identified FFPE tumors were extracted from medical archives, and tissue microarrays (TMA) were prepared depending on the quality and amount of the FFPE blocks.There was no bias in patient selection.
We require information from authors about some types of materials, experimental systems and methods used in many studies.Here, indicate whether each material, system or method listed is relevant to your study.If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.underThestudywasapprovedbytheNon-Interventional Clinical Research Ethics Committee of Hacettepe University (approval no: 2020/02-40).For in vitro experiments, sample sizes were chosen based on experience from previous experiments using similartechniques (Saatci et al.  2020, Nature Communications; Saatci et al, 2022, Cell Death & Differentiation; Mishra et al, 2018, Clinical Cancer Research)to reach statistically relevant results.Sample sizes for in vivo experiments were determined based on previous studies(Saatci et al, 2018,  Oncogene; Saatci et al. 2020, Nature Communications; Saatci et al, 2022, Cell Death & Differentiation; Mishra et al, 2018, Clinical Cancer  Research).
No blinding was performed for the other experiments since the investigators should keep careful track of protocols, most of the experiments needed multiple treatments and the samples should first be allocated into different groups.Cell Signaling, 4499) was tested by Western blot analysis of extracts from various human and mouse cell lines.Alpha-tubulin antibody (Santa Cruz, sc-32293) was tested in different human and mouse cell lines with Western blotting.ER antibody (Santa Cruz, sc-8002) was tested in different human and mouse cell lines with Western blotting.p-H2AX (S139) antibody (Santa Cruz, sc-517348) was tested in different human and mouse cell lines with Western blotting.p-Chk2 (T68) antibody (Cell Signaling, 2197) was tested by Western blot analysis of extracts from untreated or UV-treated human cells.p-Chk1 (S345) antibody (Cell Signaling, 2348) was tested by Western blot analysis of extracts from untreated or UV-treated human and mouse cells.